Oligonucleotide-based probes for detection of bacterial nucleases

Abstract

The present invention relates to a rapid detection of microbial-associated nuclease activity with chemically modified nuclease (e.g., ribonuclease) substrates, and probes and compositions useful in detection assays. Accordingly, in certain embodiments, the present invention provides a probe for detecting a microbial endonuclease comprising a substrate oligonucleotide of 2-30 nucleotides in length, a fluorescence-reporter group operably linked to the oligonucleotide, and a fluorescence-quencher group operably linked to the oligonucleotide. The fluorescence-reporter group and the fluorescence-quencher group are separated by at least one RNAse-cleavable residue, e.g., RNA base.

What is claimed is: 
     
       1. A probe for detecting a microbial endonuclease comprising an oligonucleotide of 2-30 nucleotides in length, a fluorophore operably linked to the oligonucleotide, and a quencher operably linked to the oligonucleotide, wherein the oligonucleotide comprises a chemically modified pyrimidine comprising a 2′ substituted sugar and is capable of being cleaved by a microbial nuclease but not by a mammalian nuclease. 
     
     
       2. The probe of  claim 1 , wherein the oligonucleotide is 10-15 nucleotides in length. 
     
     
       3. The probe of  claim 1 , wherein the oligonucleotide has between 0-50% purines. 
     
     
       4. The probe of  claim 1 , wherein one or more of the chemically modified pyrimidines comprises a sugar selected from the group consisting of 2′-O-methyl-ribose, 2′-O-alkyl-ribose, 2′-O-allyl-ribose, 2′-S-alkyl-ribose, 2′-S-allyl-ribose, 2′-fluoro-ribose, 2′-halo-ribose, 2′-azido-ribose, carbocyclic sugar analogue, a-anomeric sugar, epimeric sugar, arabinose, xylose, lyxose, pyranose sugar, furanose sugar, and sedoheptulose, or is a 2′-O-methyl ribonucleotide, a 2′-methoxyethoxy ribonucleotide, a 2′-O-allyl ribonucleotide, a2′-O-pentyl ribonucleotide, a 2′-O-butyl ribonucleotide, 2′-fluoro-β-D-arabinonucleotide (FANA), Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA). 
     
     
       5. The probe of  claim 1 , wherein one or more of the purines if present are chemically modified. 
     
     
       6. The probe of  claim 1 , wherein the fluorophore is selected from the group consisting of the fluorophores listed in Table 1. 
     
     
       7. The probe of  claim 1 , wherein the quencher is selected from the group consisting of the quenchers listed in Table 2. 
     
     
       8. The probe of  claim 1 , wherein the oligonucleotide is single-stranded. 
     
     
       9. The probe of  claim 1 , wherein the oligonucleotide comprises both RNA and DNA. 
     
     
       10. The probe of  claim 9 , wherein the oligonucleotide comprises a DNA di-nucleotide. 
     
     
       11. An oligonucleotide probe comprising a fluorophore operably linked to a first strand of 4-5 modified RNA nucleotides, which is operably linked to a strand of two DNA nucleotides, which is operably linked to a second strand of 4-6 modified RNA nucleotides, which is operably linked to at least one fluorescence quencher. 
     
     
       12. An oligonucleotide probe consisting of /56-FAM/mCmUmCmGTTmCmGmUmUmC/ZEN//3IAbRQSp/ (SEQ ID NO: 5). 
     
     
       13. A method of detecting a microbial infection of a sample comprising measuring fluorescence of a sample that has been contacted with a probe of  claim 1 , wherein a fluorescence level that is greater than the fluorescence level of an uninfected control indicates that the sample has a microbial infection. 
     
     
       14. The method of  claim 13 , wherein the method is performed in vivo for the detection of a microbial infection in a mammal, wherein a test fluorescence that is greater than the fluorescence level of an uninfected control indicates that the sample has a microbial infection. 
     
     
       15. The method of  claim 13 , wherein the test fluorescence level is at least 1-100% greater than the control level. 
     
     
       16. The method of  claim 13 , wherein the fluorophore is detectable at a depth of 7-14 cm in a mammal. 
     
     
       17. A method for detecting ribonuclease activity in a test sample, comprising:
 (a) contacting the test sample with a probe, thereby creating a test reaction mixture, wherein the probe comprises a nucleic acid molecule comprising:
 i. a cleavage domain comprising a single-stranded region of RNA, the single-stranded region comprising at least one internucleotide linkage and a chemically modified pyrimidine comprising a 2′ substituted sugar and; 
 ii. a fluorescence reporter group on one side of the internucleotide linkages; and 
 iii. a fluorescence-quenching group on the other side of the internucleotide linkages; 
 
 (b) incubating the test reaction mixture for a time sufficient for cleavage of the probe by a ribonuclease in the sample; and 
 (c) determining whether a detectable fluorescence signal is emitted from the test reaction mixture, wherein emission of a fluorescence signal from the reaction mixture indicates that the sample contains ribonuclease activity. 
 
     
     
       18. The method of  claim 17 , further comprising:
 (d) contacting a control sample with the probe, the control sample comprising a predetermined amount of ribonuclease, thereby creating a control reaction mixture; 
 (e) incubating the control reaction mixture for a time sufficient for cleavage of the probe by a ribonuclease in the control sample; and 
 (f) determining whether a detectable fluorescence signal is emitted from the control reaction mixture; wherein detection of a greater fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains greater ribonuclease activity than in the control sample, and wherein detection of a lesser fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains less ribonuclease activity than in the control sample. 
 
     
     
       19. The method of  claim 18 , wherein the predetermined amount of ribonuclease is no ribonuclease, such that detection of a greater fluorescence signal in the test reaction mixture than in the control reaction mixture indicates that the test sample contains ribonuclease activity. 
     
     
       20. The method of  claim 17 , further comprising contacting the test sample with a buffer before or during step (a). 
     
     
       21. The method of  claim 17 , wherein the chemically modified pyrimidine comprises a sugar selected from the group consisting of 2′-O-methyl-ribose, 2′-O-alkyl-ribose, 2′-O-allyl-ribose, 2′-S-alkyl-ribose, 2′-S-allyl-ribose, 2′-fluoro-ribose, 2′-halo-ribose, 2′-azido-ribose, carbocyclic sugar analogue, a-anomeric sugar, epimeric sugar, arabinose, xylose, lyxose, pyranose sugar, furanose sugar, and sedoheptulose.